B6C3-Tg(tetO/Prnp-TARDBP*)2Vle/J

Stock number: 028590
Product name: tetO-208 TDP-43 transgenic line #2
Research areas: Neurobiology Research, Research Tools

Description

TetO-208 TDP-43 transgenic line 2 mice have a Tet-responsive element and mouse prion protein promoter sequences directing expression of a human TDP-43 C-terminal fragment (amino acids 208-414), or 208 TDP-43 CTF, that is associated with frontotemporal lobar degeneration with TDP-43 pathology in human brain. These mice may be used to generate Tet-Off/Tet-On mutant animals with conditional (inducible/reversible) expression of 208 TDP-43 CTF.

Detailed description

The tetO-208 TDP-43 transgene has the Tet-responsive element (TRE or tetO) and mouse prion protein promoter sequences (PrP or Prnp) directing expression of amino acids 208-414 from human TDP-43 (TARDBP). Accumulation of the TDP-43 C-terminal fragment (208 TDP-43 CTF) is associated with frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) in human brain. When these tetO-208 TDP-43 transgenic line 2 mice are exposed to tetracycline-controlled transactivator protein (tTA) or reverse tetracycline-controlled transactivator protein (rtTA), 208 TDP-43 CTF expression in the resulting animals can be regulated with tetracycline or its analog doxycycline (dox).

Breeding the B6C3F1 tetO-208 TDP-43 mice to C57BL/6J-congenic mice with forebrain-specific tTA expression (from Stock No. 003010) results in bigenic Camk2a-tTA/tetO-208 TDP-43 animals (CAMK-208 TDP-43 Tg). Following dox withdrawal, those CAMK-208 TDP-43 Tg mice exhibit highly-insoluble TDP-43 CTF accumulation in neuronal cytoplasm and dendrites, without formation of the large cytoplasmic TDP-43 inclusions seen in FTLD-TDP and ALS tissue. Hippocampus showed progressive neurodegeneration and astrogliosis in the dentate gyrus (DG), accompanied by phosphorylated TDP-43 in the CA1 subfield and ubiquitin and mitochondria accumulations in the stratum lacunosum moleculare (SLM) layer. The observed neurodegeneration was without changes in the level, solubility or subcellular distribution of nuclear endogenous full-length TDP-43, and was also independent of TDP-43 phosphorylation. After 13 months of transgene expression (off-dox), resumption of dox-treatment led to rapid clearance of 208 TDP-43 CTF and phosphorylated TDP-43, which ameliorated neuron loss in the DG despite persistence of ubiquitin accumulation in the SLM.
In addition, breeding B6C3F1 tetO-208 TDP-43 to NEFH-tTA line 8 mice (broad tTA expression in brain but only modest levels in DG granular cells; see Stock No. 025397) results in bigenic NEFH-tTA/tetO-208 TDP-43 animals (NEFH-208 TDP-43 Tg). Following dox withdrawal, NEFH-208 TDP-43 Tg exhibit similarities to CAMK-208 TDP-43 Tg - including high levels of insoluble 208 TDP-43 CTF in the cortex and hippocampus, with accumulation of p409/410 TDP-43 predominantly in the CA1 region and ubiquitin in the SLM region of the hippocampus. Uniquely, NEFH-208 TDP-43 Tg show no overt loss of DG neurons up to 19 months (as <5% of DG cells expressed 208 TDP-43 CTF).

The donating investigator reports that hemizygous tetO-208 TDP-43 transgenic mice are viable and fertile with no observed phenotype defects or behavioral abnormalities, and they have not tried to generate homozygous mice to date (September 2016). The transgene insertion site(s) is not known. Multiple copies are likely, however, as the inducible expression level of TDP-43_208-414 in hemizygous mice is 3-6-fold greater than endogenous TDP-43 levels.

Note on tetO/Prp promoter: While the untranslated sequences from Prnp have been shown in other mouse models to result in minimal levels of transgene expression in the brain before Tet-induction (see Stock Nos. 025104/027783), it has not yet been determined if these tetO-208 TDP-43 transgenic line 2 mice exhibit any such expression before Tet-induction (September 2016).

Development

The tetO-208 TDP-43 transgenic line 2 mice were created in the laboratory of Dr. Virginia Man-Yee Lee (University of Pennsylvania). The tetO-208 TDP-43 transgene has the Tet-responsive element and mouse prion protein non-coding exons 1-2 upstream of a cDNA sequence encoding amino acids 208-414 of human TAR DNA binding protein (TARDBP or TDP-43) followed by the mouse prion protein non-coding exon 3 and 3' UTR of the moPrP.XhoI vector (including polyadenylation signal).

The Tet-responsive element (TRE) contains copies of the 42-bp tet operator sequence (tetO) followed by the constitutively active, minimal human cytomegalovirus promoter (P_minCMV). The tetO-208 TDP-43 transgene used TRE (from pTetSplice bp 1-318) and mouse prion protein (PrP or Prnp) sequences from the moPrP-tetP vector (a modified version of moPrP.XhoI).

The ~6.7 kbp transgene was injected into the pronucleus of fertilized eggs from (C57BL/6J x C3HeJ)F1 matings (B6C3F1/J ; Stock No. 100010). Founder animals were bred to B6C3F1/J for germline transmission. Three founder lines were generated. Founder line 2 had the highest inducible expression of 208 TDP-43 C-terminal fragments (CTFs), and all three lines had the same inducible 208 TDP-43 CTF distribution and biochemical properties.

The tetO-208 TDP-43 transgenic line 2 was then maintained by breeding hemizygous mice with B6C3F1/Crl for several generations, and then hemizygous males (black or agouti) were sent to The Jackson Laboratory Repository in 2016.
Upon arrival, sperm was cryopreserved. To establish our living colony, an aliquot of the frozen sperm was used to fertilize oocytes from B6C3F1/J females (Stock No. 100010). After that, our live colony will be maintained by breeding hemizygous mice with wildtype (noncarrier) mice from the colony, and animals will be selected to have the C57BL/6-derived Pde6b allele (rather than the C3H-derived Pde6b^rd1). Our colony will also be periodically bred to B6C3F1/J mice (Stock No. 100010) to maintain ~50/50% mix of C57BL/6 and C3H.


The donating investigator reports that the transgene insertion site(s) is not known. Multiple copies are likely, however, as the inducible expression level of TDP-43_208-414 in hemizygous mice is 3-6-fold greater than endogenous TDP-43 levels.

Allele

Symbol: Tg(tetO/Prnp-TARDBP*)2Vle
Name: transgene insertion 2, Virginia M Y Lee
MGI accession ID: MGI:5792270
Generation method: Transgenic
Attributes: Inducible; Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(tetO/Prnp-TARDBP*)2Vle
Marker name: transgene insertion 2, Virginia M Y Lee
Chromosome: UN
Strain of origin: (C57BL/6J x C3H/HeJ)F1
Expressed genes: TARDBP,TAR DNA binding protein,human
Site of expression: Primarily in the cortex, hippocampus and olfactory bulb of the brain.
Molecular note: The transgenic construct contains the Tet-responsive element and mouse prion protein non-coding exons 1-2 upstream of a cDNA sequence encoding amino acids 208-414 of human TAR DNA binding protein (TARDBP or TDP-43) followed by the mouse prion protein non-coding exon 3 and 3' UTR of the moPrP.XhoI vector (including polyadenylation signal). Three founder lines were generated. Founder line 2 had the highest inducible expression of 208 TDP-43 C-terminal fragments (CTFs), and all three lines have the same inducible 208 TDP-43 CTF distribution and biochemical properties.